Primer composition and application thereof in detection of animal bifidobacterium subsp. Lactis HN019

Abstract

The invention discloses a primer composition and application of the primer composition in detection of animal bifidobacterium subsp. Lactis HN019. According to the present invention, bifidobacterium animalis subsp. Lactis HN019 is adopted as an object, bacterial strain specific genetic markers are screened through whole genome sequencing and single nucleotide polymorphism analysis, and a fluorescent quantitative PCR technology is combined so as to obtain the efficient, specific and stable HN019 quantitative detection primer composition and the method thereof; through system verification, the method is suitable for an HN019 strain level detection technical framework in scientific research and industrial application scenes, and also provides key technical support for market supervision and industrial healthy development of probiotic foods.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology biological detection, and in particular relates to a primer composition and an application thereof in detecting animal Bifidobacterium lactis subspecies HN019. Background Art

[0002] Probiotics can exert health benefits in the gastrointestinal tract by regulating the host's microecological balance and have become an important part of modern disease prevention and treatment strategies. Among the many probiotic strains, Bifidobacterium, especially Bifidobacterium animalis subsp. lactis HN019 (B. animalis subsp. lactis HN019), stands out for its excellent environmental adaptability, biological stability and significant health benefits, and its application value in the early stages of life is particularly outstanding. This strain is widely added to infant formula, dietary supplements and dairy products. It can effectively reduce the incidence of diarrhea by competitively excluding pathogenic microorganisms and maintaining the integrity of the intestinal mucosal barrier. In addition to its gastrointestinal protective effects, HN019 can also have a positive impact on the overall health of the body by regulating immune metabolic pathways, inhibiting inflammatory responses, and reducing pathological damage.

[0003] The efficacy and safety of probiotics are highly dependent on strain-specific traits. In the booming probiotics industry, accurate strain-level identification and quantitative analysis are core technical requirements for product development, quality control, and compliance assessment. However, existing detection methods face significant technical bottlenecks: traditional culture methods based on morphological and biochemical characteristics are time-consuming and labor-intensive, and the resolution is insufficient, making it difficult to meet the needs of modern high-throughput detection; although traditional molecular typing technologies such as pulsed-field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) can achieve strain identification, they have problems such as complex operation, strong equipment dependence, and limited large-scale application. In recent years, breakthroughs in whole-genome sequencing (WGS) technology have provided an innovative solution for high-resolution strain detection. Methods based on whole-genome single nucleotide polymorphism (SNP) analysis can achieve detailed analysis of genetic differences between strains, providing strong support for specific identification. Further combining WGS-SNP analysis with fluorescent quantitative PCR (qPCR) technology can construct a standardized detection system suitable for industrial-grade quality control and regulatory supervision. However, there is currently no efficient, specific, simple and stable HN019 detection method based on whole genome sequencing (WGS) and single nucleotide polymorphism (SNP) analysis. Summary of the Invention

[0004] In order to make up for the shortcomings of the existing technology, the present invention takes animal Bifidobacterium lactis subsp. lactis HN019 as the object, screens strain-specific genetic markers through whole genome sequencing and single nucleotide polymorphism analysis, and combines fluorescent quantitative PCR technology to develop an efficient, specific and stable quantitative detection method for HN019.

[0005] In one aspect, the present invention provides a primer composition, comprising a forward primer and a reverse primer, wherein the forward primer comprises a nucleotide sequence as shown in SEQ ID NO: 4, and the reverse primer comprises a nucleotide sequence as shown in SEQ ID NO: 5.

[0006] In one or more embodiments, the primer composition further comprises a probe.

[0007] Preferably, the probe is modified with a fluorescent group, and the fluorescent group is selected from one or more of FAM, TET, JOE, HEX, CY3, TAMRA, ROX, Taxa Red, LC RED640, CY5, LC RED705, AMC and Quasar.

[0008] More preferably, the fluorescent group is FAM.

[0009] Preferably, the probe has a minor groove binder modification.

[0010] In one or more embodiments, the probe comprises the nucleotide sequence shown in SEQ ID NO:6.

[0011] In another aspect, the present invention provides use of the primer composition according to any embodiment herein for quantitatively detecting Bifidobacterium animalis subsp. lactis HN019 or its DNA in a sample.

[0012] In another aspect, the present invention provides a method for quantitatively detecting animal Bifidobacterium lactis subsp. lactis HN019 or its DNA in a sample, the method comprising using the primer composition described in any embodiment herein to perform a nucleic acid amplification reaction on the sample to specifically amplify the target DNA fragment of animal Bifidobacterium lactis subsp. lactis HN019.

[0013] In another aspect, the present invention provides a method for screening a primer composition for quantitatively detecting Bifidobacterium animalis subsp. lactis HN019 or its DNA, the method comprising the following steps:

[0014] S1: comparing the genome of Bifidobacterium animalis subsp. lactis HN019 with the genomes of one or more other Bifidobacteria to screen for specific SNP sites in the genome of Bifidobacterium animalis subsp. lactis HN019;

[0015] S2: Using the sequence near the specific SNP site in the genome of Bifidobacterium animalis subsp. lactis HN019 as the target fragment sequence, the primer combination was designed using software;

[0016] S3: Synthesize the designed primer combination, use it to perform nucleic acid amplification reaction on the sample, and screen out the primer combination that can specifically amplify the target fragment sequence of animal Bifidobacterium lactis subsp. lactis HN019; wherein the sample includes animal Bifidobacterium lactis subsp. lactis HN019 or its DNA, and one or more other Bifidobacteria or their DNA.

[0017] Preferably, the sample is a sample of food, beverage, medicine, health product, cosmetic or skin care product.

[0018] Preferably, the sample is a sample of air, water or soil.

[0019] Preferably, the sample is a sample of a composite bacterial agent.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The primer combination provided by this invention can be used for efficient, specific, simple, and stable quantitative detection of HN019 in HN019 DNA samples, HN019 bacterial suspension samples, or probiotic products containing HN019. Through systematic validation of the method's specificity, sensitivity, repeatability, and reproducibility, a technical framework for HN019 strain-level detection suitable for both scientific research and industrial applications has been established, providing key technical support for market regulation and the healthy development of the probiotic food industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the primer-specific detection result of Bifidobacterium animalis subsp. lactis HN019 strain.

[0023] Figure 2 This is the fluorescence value-Cq graph of qPCR of HN019 DNA with gradient concentrations.

[0024] Figure 3 The standard curve of primer combination detection HN019 DNA concentration-Cq value.

[0025] Figure 4 This is the fluorescence value-Cq graph of qPCR of HN019 bacterial solution with gradient concentrations.

[0026] Figure 5 This is the primer combination detection HN019 bacterial liquid concentration-Cq value standard curve.

[0027] Figure 6 This is a graph showing the HN019 strain content in probiotic products containing the HN019 strain. DETAILED DESCRIPTION

[0028] Example 1: Single Nucleotide Polymorphism (SNP) Analysis

[0029] The full genome sequences of the 31 strains of Bifidobacterium animalis subsp. lactis listed in Table 1 were obtained from the NCBI database. Sequence alignment analysis was performed using the HN019 genome as the reference sequence using MUMmer 3.23 software. First, a global pairwise alignment of the 30 test strains with the HN019 genome was performed using the nucmer tool to generate a genomic similarity and difference map. Low-confidence alignment regions and redundant sequences were then removed using the delta-filter program to retain high-quality alignment data. Finally, the show-snp tool was used to identify SNP variants between HN019 and the other 30 strains. Correlation analysis was performed by Shanghai Meiji Biopharmaceutical Technology Co., Ltd.

[0030] Table 1: Genomic information of 31 strains of Bifidobacterium animalis subsp. lactis

[0031]

[0032]

[0033] Using the full genome sequence of strain HN019 as a reference, the full genome sequence of 30 other strains was compared. Six SNPs were identified among the 10,357 SNPs in the HN019 genome, relative to the other 30 strains. Four of these SNPs were located in single-copy genes. Three sets of primers and probes were designed using Primer Express 3.0 software, selecting a specific SNP located in the galK gene. The four SNPs were selected based on probe alignment with the amplified fragment and inclusion of the specific site. Optimization was also performed based on parameters such as Tm value, primer end stability, GC content, and secondary structure. Primers and probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The sequences of the primer combinations (primers and probes) are shown in Table 2 below. The probes contain a FAM (5'-carboxyfluorescein) modification at the 5' end and an MGB (minor groove binder) modification at the 3' end.

[0034] Table 2: Information on gene fragments to be amplified and their primer combinations

[0035]

[0036]

[0037] Example 2: Primer Composition Specificity Detection

[0038] DNA was extracted from 20 different Bifidobacterium strains (including commercial and standard strains) listed in Table 3 using a bacterial genomic DNA extraction kit (purchased from TIANGEN, Catalog No. DP302). The extracted DNA samples were subjected to 1.0% agarose gel electrophoresis and recovered. DNA concentration, 260 / 280 ratio, and 260 / 230 ratio were measured using a DeNovix DS-11 microspectrophotometer. The recovered DNA was used as a DNA template and stored at -20°C until use.

[0039] Table 3: Information on strains used in primer combination specificity studies

[0040]

[0041] The primer specificity of the DNA templates of 20 Bifidobacterium strains was studied. The three sets of primer compositions in Table 2 were used to prepare the following qPCR reaction system for reaction: 2×Takara Prober Taq mix 10μL, forward primer (5μM) 1μL, reverse primer (5μM) 1μL, probe (10μM) 1μL, DNA template (50ng / μL) 1μL, deionized water was added to 20μL. The reaction procedure is: the first stage is 95℃, 2min; the second stage is 95℃, 5sec, 64℃, 35sec, for a total of 40 cycles. Each group of experiments was repeated three times in parallel, and sterile water was used as a blank control. During the qPCR process, fluorescence with a wavelength of 465-510nm was detected. The specific amplification of the same subspecies strain by the second group of primer compositions is significantly better than that of the first and third groups of primer compositions. The results of the second group are shown in Figure 2. Figure 1 As shown, the curve of "HN019" corresponds to the test results of HN019, NC corresponds to the test results of 19 strains other than HN019 in Table 3, and BC corresponds to the test results of the blank control.

[0042] Depend on Figure 1 As shown, the second primer combination, designed using a SNP located in the galK gene, can effectively distinguish HN019 from 19 other strains of Bifidobacterium through qPCR amplification. Using the second primer combination, the Cq values ​​for three replicates corresponding to the HN019 strain were 19.25, 19.27, and 19.07, respectively. No significant amplification was observed for the other strains or the blank control. According to general criteria, a Cq value less than 35 is considered positive. Furthermore, the second primer combination demonstrated significantly better specific amplification for strains of the same subspecies than the first and third primer combinations. These results demonstrate that the second primer combination in Table 2 has outstanding specificity for detecting HN019 DNA.

[0043] Example 3: Assay stability during passage

[0044] HN019 was cultured and passed to the 35th generation, and the bacterial suspensions of the 5th, 15th, 25th and 35th generations were collected (concentration 10 8 CFU / mL), and DNA templates were obtained for each generation according to the method in Example 2. qPCR systems were prepared for amplification, using the primers and probes from Group 2 in Table 2. Each experiment was repeated in triplicate, with sterile water as a blank control. The results of the Cq value analysis for each group are shown in Table 4:

[0045] Table 4: Specific amplification and passage stability test results

[0046]

[0047] As shown in Table 4, the standard deviation of the Cq value of the specific detection of the HN019 strain from the 5th to the 35th generation was 0.24, and the relative standard deviation was 1.31%, indicating that the preferred second set of primers designed for the SNP site on the galK gene has good stability during the HN019 passage process and can be used for the detection of HN019 strains of limited generations.

[0048] Example 4: Sensitivity of primer combinations for detecting HN019 DNA and HN019 bacterial suspension

[0049] (1) Sensitivity of detecting HN019 DNA

[0050] The HN019 DNA obtained in Example 2 was prepared into a 50 ng / μL solution, and then diluted 10-fold to obtain HN019 DNA solutions with gradient concentrations (0.0005 ng / μL, 0.005 ng / μL, 0.05 ng / μL, 0.5 ng / μL, 5 ng / μL, and 50 ng / μL), which were used to prepare PCR systems. qPCR amplification was performed using the second set of primer combinations in Table 2. The fluorescence changes during the qPCR process were detected, and a standard curve was plotted with the logarithm of the DNA concentration as the X-axis and the average Cq value as the Y-axis. The fluorescence changes were as follows: Figure 2 The standard curve is shown as Figure 3 shown.

[0051] Depend on Figure 2 It can be seen that there is a good linear relationship between DNA concentration and fluorescence value. Figure 3 Available, Figure 3 The slope of the standard curve is -3.311, R 2The calculated amplification efficiency was 100.46%, and the DNA concentration detection limit (Cq ≤ 35) of this amplification system was approximately 0.0005 ng / μL. These results demonstrate that this primer combination has high sensitivity for detecting HN019 DNA and good DNA concentration linearity.

[0052] (2) Sensitivity of detecting HN019 bacterial solution

[0053] In order to meet the detection needs of dairy companies for the number of lactic acid bacteria colonies, the quantitative results are linked to the bacterial colony-forming units and the concentration of the bacterial solution is directly detected. In this example, the second group of primer combinations in Table 2 is used to detect HN019 bacterial solution and the sensitivity of the detection is determined.

[0054] Prepare 1 mL of 10 8 ~10 3 CFU / mL gradient concentration (10 3 CFU / mL, 10 4 CFU / mL, 10 5 CFU / mL, 10 6 CFU / mL, 10 7 CFU / mL and 10 8 CFU / mL) of HN019 strain culture fluid was used to extract DNA according to the method of Example 2, and the qPCR system was prepared with the second set of primer combinations in Table 2 for amplification; each group of experiments was repeated three times, and sterile water was used as a blank control. The fluorescence changes during the qPCR process were detected, and a standard curve was drawn with the logarithm of the bacterial concentration as the X-axis and the average Cq value as the Y-axis. Figure 4 The standard curve is shown as Figure 5 shown.

[0055] Depend on Figure 4 It can be seen that there is a good linear relationship between bacterial concentration and fluorescence value. Figure 5 It can be seen that when the bacterial concentration is greater than or equal to 10 3 CFU / mL, the test results were all positive (Cq value < 35); the slope value of the standard curve was -3.370, R 2 The sensitivity of the primer combination to detect HN019 bacterial solution was high, and the detection results had good linearity in bacterial solution concentration.

[0056] Example 5: Repeatability and reproducibility verification

[0057] The repeatability of the selected primer combination for detecting HN019 was verified by repeating the experiment in a short period of time (one week between the two experiments). The reproducibility of the selected primer combination for detecting HN019 was verified by testing on two different qPCR platforms (Roche Light Cycler 480 and ABI7500). Repeatability and reproducibility tests were performed at three different DNA concentrations (0.5 ng / μL, 5 ng / μL, and 50 ng / μL). Each experiment was repeated in triplicate, with sterile water as a blank control. The results are shown in Tables 5 and 6:

[0058] Table 5: Amplification reproducibility verification

[0059]

[0060]

[0061] Table 6: Amplification reproducibility verification

[0062]

[0063] As shown in Tables 5 and 6, the RSDs for the repeatability validation results for the interval time test ranged from 1.12% to 2.21%. The RSDs for the reproducibility test results between two different qPCR platforms (Roche Light Cycler 480 and ABI 7500) ranged from 1.55% to 2.65%, both significantly below the qPCR RSD requirement of less than 25%. Therefore, this method demonstrated excellent repeatability and reproducibility.

[0064] Example 6: Primer Composition for Detecting HN019 and Its Content in Probiotic Products

[0065] The second set of primer compositions in Table 2 was used to quantitatively detect the HN019 strain in five batches of probiotic powder samples provided by the company. The powder consisted of HN019, Lactobacillus rhamnosus GG, Lactobacillus acidophilus NCFM, and excipients such as oligosaccharides. The samples were prepared according to the method of the Chinese national standard (GB 4789.35-2016).

[0066] Dissolve 25g of sample (mass is recorded as A) in 225mL (volume is recorded as B) of sterile saline, then perform 10-fold gradient dilution and record the dilution multiple (dilution multiple is recorded as C); 9 The HN019 bacterial suspension with a CFU / mL was serially diluted 10-fold to prepare 10 4 ~10 8 CFU / mL bacterial solution with gradient concentration.

[0067] DNA was extracted from each sample dilution and each HN019 bacterial suspension dilution according to the method of Example 2, and a qPCR system was prepared using the second set of primer compositions in Table 2, and qPCR amplification was performed on a Roche Light Cycler 480 platform.

[0068] For each dilution of HN019 bacterial suspension, the following Figure 5 The standard curve shown is a logarithm of bacterial concentration (CFU / mL) as the X axis and Cq value as the Y axis. The formula of the standard curve is Y = -3.347 × X + 44.17, R 2 =0.9944.

[0069] For each sample dilution, the content of HN019 in the sample was calculated according to the following formula. The calculation results of the content of each batch of samples are as follows: Figure 6 As shown;

[0070] M=10 x ×(A+B) / A×C;

[0071] M is the HN019 content in the probiotic powder (CFU / g); X is the logarithm of the bacterial concentration, which is calculated by substituting the Cq value of each sample dilution into the formula of the above standard curve; A is the weighed mass of the test sample (g); B is the volume of the dilution (mL); and C is the dilution factor of the sample solution.

[0072] Depend on Figure 6 Using the standard curve and the above formula, the HN019 strain content in the five batches of samples was calculated to be between 11.91 ± 0.03 and 12.11 ± 0.12 Log (CFU / g). This primer combination can also be used to detect HN019 in composite probiotic powder samples containing large amounts of oligosaccharides and excipients.

Claims

1. A primer composition, characterized in that The primer composition comprises a forward primer and a reverse primer, wherein the forward primer comprises a nucleotide sequence as shown in SEQ ID NO: 4, and the reverse primer comprises a nucleotide sequence as shown in SEQ ID NO:

5.

2. The primer composition according to claim 1, wherein The primer composition also includes a probe.

3. The primer composition according to claim 2, wherein The probe includes the nucleotide sequence shown in SEQ ID NO:

6.

4. Use of the primer combination as claimed in any one of claims 1 to 3 in the quantitative detection of Bifidobacterium animalis subsp. lactis HN019 or its DNA in a sample.

5. The use according to claim 4, characterized in that The sample is a sample of a composite bacterial agent.

6. A method for quantitatively detecting Bifidobacterium animalis subsp. lactis HN019 or its DNA in a sample, the method comprising performing a nucleic acid amplification reaction on the sample using the primer composition according to any one of claims 1 to 3 to specifically amplify a target DNA fragment of Bifidobacterium animalis subsp. lactis HN019.

7. The method according to claim 6, wherein The sample is a sample of a composite bacterial agent.

8. A method for screening a primer composition for quantitatively detecting animal Bifidobacterium lactis subsp. lactis HN019 or its DNA, characterized in that: The method comprises the following steps: S1: comparing the genome of Bifidobacterium animalis subsp. lactis HN019 with the genomes of one or more other Bifidobacteria to screen for specific SNP sites in the genome of Bifidobacterium animalis subsp. lactis HN019; S2: Using the sequence near the specific SNP site in the genome of Bifidobacterium animalis subsp. lactis HN019 as the target fragment sequence, the primer combination was designed using software; S3: Synthesize the designed primer combination, use it to perform nucleic acid amplification reaction on the sample, and screen out the primer combination that can specifically amplify the target fragment sequence of animal Bifidobacterium lactis subsp. lactis HN019; wherein the sample includes animal Bifidobacterium lactis subsp. lactis HN019 or its DNA, and one or more other Bifidobacteria or their DNA.

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